Immunoregulatory effects of collagen hydrolysate from Asian bullfrog (Rana tigerina) skin in macrophages: Impact of simulated digestion on bioactivity

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In this study, collagen hydrolysate (CH) was prepared from Asian bullfrog skin using papain hydrolysis combined with ultrasound-assisted processing, and it exhibited notable immune-enhancing and anti-inflammatory properties in its undigested form. However, gastrointestinal (GI) digestion differentially influences these bioactivities. While the immune-stimulatory activity of digested CH (d-CH) is significantly reduced following digestion, its anti-inflammatory effects remain largely preserved. Structural profiling using size exclusion chromatography (SEC) with refractive index (RI) and ultraviolet (UV) detection revealed a heterogeneous distribution of collagen hydrolysate fractions in undigested CH, ranging from 12.7 to 117 kDa. Following simulated GI digestion, CH underwent extensive molecular breakdown, leading to the disappearance of higher-molecular-weight species and the emergence of a single, dominant low-molecular-weight fraction (∼4.2 kDa), indicative of a more uniform collagen-derived fraction composition and potentially improved accessibility for absorption. In an LPS-induced macrophage inflammation model, d-CH effectively suppressed key pro-inflammatory mediators, including nitric oxide (NO), COX-2, iNOS, IL-1β, IL-6, and TNF-α, while significantly upregulating the anti-inflammatory cytokine IL-10. Additionally, d-CH inhibited the activation of pivotal inflammatory signaling cascades such as NF-κB and MAPKs. Collectively, these findings underscore the importance of structural transformation during digestion in shaping the functional properties of CH and support its potential as a bioactive ingredient in functional foods or nutraceuticals aimed at mitigating inflammation-related disorders.